Liver failure remains a devastating syndrome resulting from the loss of hepatic cell mass below a critical level. Although the prognosis of patients is greatly improved by orthotopic liver transplantation, treatment is limited by worldwide shortages of donor organs. In order to overcome these problems, alternative approaches, such as bio-artificial liver devices, albumin dialysis and cellular based therapy are being evaluated. In recent years, the feasibility to repopulate the liver with different cell types, such as mature and fetal hepatocytes, embryonic stem cells, intrahepatic progenitor cells and bone marrow derived cells, have been assessed in various animal models of liver disease.
Liver Development
Mouse embryonic and fetal liver development can be divided into different consecutive steps. During gastrulation (ED6-ED6.5), future definitive endodermal and mesodermal cells migrate through the primitive streak, located at the prospective posterior and proximal-lateral pole of the embryo. First, anterior endodermal cells ingress the primitive streak, migrate towards the distal tip of the epiblast cup and displace the visceral endoderm. The mesoderm migrates between the epiblast and endoderm. Definitive endoderm is characterized by the transient expression of primitive streak markers (LHX1, MIXL1, WNT3, LHX1, brachyury) and CXCR4, Sox17, HNF3b, Goosecoid and E-Cadherin. In contrast, primitive endoderm (visceral and parietal endoderm), which gives rise to the yolk sac, expresses Sox17, Sox7, and HNF3B. After gastrulation, embryonic progenitors of the digestive and respiratory organs initially exist in a single cell thick, epithelial sheet of endoderm that lines the ventral surface of the embryo. Then, the endoderm folds into a gut tube to form the foregut, midgut and hindgut endoderm. At ED8.25, ventral foregut is guided towards a hepatic fate under the influence of cytokines secreted by the adjacent cardiac mesoderm (aFGF-bFGF) and septum transversum mesenchyme (BMPs). After this specification (ED0.5-ED10), the resident cells of the primitive liver bud, consisting of bipotential hepatoblasts, undergo balanced events including proliferation, apoptosis, and differentiation to eventually constitute a functioning organ. This further maturation occurs through fibroblast growth factors (aFGF-FGF4-FGF8), Wnt signaling, factors secreted by the invading endothelial cells, the transiently (ED10) present hematopoietic cells in the fetal liver (Oncostatin M) and from the surrounding non-parenchymal cells (HGF). At ED14, bipotential hepatoblast become either fully mature hepatocytes or cholangiocytes. This determination depends upon the TGBβ/Activin and Notch2/Jagged1 signaling pathway.
Hepatic Stellate Cells (HSC):
reside in perisinusoidal recesses between adjacent hepatocytes and represent ±8% of the liver. They project long processes (˜50 μm) in the space of Disse and between adjacent hepatocytes. In the adult liver, HSC are quiescent, produce small amounts of ECM and store vitamin A in the form of retinyl esters in lipid droplets. HSC secrete many cytokines and virtually all growth factors. Not only do the cells secrete the cytokines, they also respond to them. Under pathological conditions, HSC are activated to become myofibroblast-like cells. They proliferate, acquire contractile properties, lose their capacity to store vitamin A and produce excessive amounts of ECM, causing scar formation. This activation is accompanied by changes in gene and miRNA expression, phenotype and function. If the liver injury is limited, HSC revert to a quiescent state or die by apoptosis. However, after prolonged injury, HSC become resistant to apoptosis and their activated phenotype may not be reversible.
HSC can be isolated from normal livers by taking advantage of their high content of lipid droplets that are rich in vitamin A. These lipid droplets are fluorescent under UV light allowing isolation of quiescent HSC by FACS. Moreover, due to their low density it is also possible to isolate them by gradient centrifugation. HSC isolated from normal livers have a quiescent phenotype showing a limited proliferative capacity in culture. Culture conditions for maintaining the quiescent phenotype for an extensive period of time are not known. After 24-48 hours in culture, the cells acquire most of the phenotypic and functional characteristics of activated HSC in injured liver, although their phenotype is not completely identical. When cultured under activating conditions human HSC can be expanded up to 20 doublings.
Liver fibrosis, which is the outcome of persistent hepatic inflammation, if left unmanaged, has serious long-term consequences for patient morbidity and mortality. Antifibrotic therapies must be aimed at inhibiting the activated hepatic stellate cell, which is responsible for the fibrotic response to injury. Development of specific anti liver-fibrosis drugs is, however, hampered by the fact that large numbers of quiescent human hepatic stellate cells, the chief cell responsible for liver fibrosis, are not available, as most isolated cell populations are culture-expanded, during which they quickly acquire an activated cell phenotype (Friedman et al., J Biol Chem 264:10756-10762 (1989); Friedman S. L., Hepatology 40:1041-1043 (2004)).
Hepatic sinusoidal endothelial cells (HSEC) comprise 20% of all liver cells and are strongly fenestrated cells. HSEC use these fenestrae (75-250 nm) to exclude larger particles and cells from the space of Disse but can also eliminate soluble macromolecules and colloidal particles from blood by active scavenging via specific endocytic receptors. The basement membrane underneath HSEC differs from other endothelia in that basement matrix molecules are present but not organized in a structure visible by transmission EM. HSEC, together with HSC, control the vascular tone in the sinusoidal capillaries. Under pathological conditions, e.g. in cirrhosis, HSEC undergo dramatic changes (collectively called ‘capillarisation’): the fenestrated sieve-like endothelium is replaced by a classical closed capillary endothelium, with a dense basal lamina visible by EM. This results in reduced access of hepatocytes to O2 and nutrients. Also, upon ageing, the HSEC undergo phenotypical changes, called ‘pseudo-capillarisation’, characterized by defenestration, basement membrane deposition and functional deterioration.
Aside from HSEC, liver also contains non-sinusoidal endothelium (in hepatic arteries and veins) and the lymphatic endothelium. Each of these can be distinguished by a separate combination of surface markers. The three major HSEC endocytosis receptors are among the group of unique and specific markers of these cells: i) Stab-2=the major scavenger receptor of HSEC22; ii) CD32b=SE-1=the unique FcγIIb receptor, the only Fc-receptor that mediates endocytosis, is present (in liver) only in HSEC; iii) the mannose receptor, clearing blood borne collagen alpha chains (=denatured collagen of several collagen types) and macromolecules and colloids that carry terminal mannose, ManNAc or GlcNAc residues. These receptors can be structurally identified by specific immune staining (live and fixed cells), or functionally by exposing intact cells to ligands for these three endocytosis receptors, which will accumulate the ligands only in HSEC. In addition, analogous to hepatocytes, within the sinusoidal endothelial population, differences have been detected in morphological and functional characteristics, depending on the location within the sinusoid (‘zonation’): HSEC from the periportal region are less fenestrated, have a low cytoplasmic porosity index and efficiently bind wheat germ agglutinin (WGA), while those in the perivenous region are highly fenestrated, have a high cytoplasmic porosity index and only weakly bind WGA.
As is true for hepatocytes and HSC, culture of HSEC leads very quickly to de-differentiation and/or activation, whereby many functional attributes from the cells are lost. For instance, HSEC lose their endocytic ability after only a few hours of culture. This rapid loss of the “signature” scavenger function of HSEC in vitro may be significantly counteracted by specially designed culture media and physiological O2 tension. In addition, co-culturing of hepatocytes, HSEC and HSC has proven fruitful in maintenance of their “in vivo” phenotype.